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- PDB-9pcx: 22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), hydroly... -

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Basic information

Entry
Database: PDB / ID: 9pcx
Title22bin20S complex (NSF-alphaSNAP-2:2 syntaxin-1a:SNAP-25), hydrolyzing, class 14
Components
  • Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera
  • Syntaxin-1A
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / ATPase / SNARE / hydrolysis / disassembly / translocation / exocytosis / neurotransmitter release / synapse / synaptic transmission / membrane fusion
Function / homology
Function and homology information


BLOC-1 complex / myosin head/neck binding / synaptic vesicle fusion to presynaptic active zone membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / presynaptic dense core vesicle exocytosis / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / extrinsic component of presynaptic membrane / calcium ion-regulated exocytosis of neurotransmitter ...BLOC-1 complex / myosin head/neck binding / synaptic vesicle fusion to presynaptic active zone membrane / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / presynaptic dense core vesicle exocytosis / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / extrinsic component of presynaptic membrane / calcium ion-regulated exocytosis of neurotransmitter / Glutamate Neurotransmitter Release Cycle / Norepinephrine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / positive regulation of norepinephrine secretion / positive regulation of catecholamine secretion / regulated exocytosis / Dopamine Neurotransmitter Release Cycle / synaptic vesicle docking / SNARE complex disassembly / regulation of synaptic vesicle priming / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / regulation of establishment of protein localization / positive regulation of calcium ion-dependent exocytosis / ribbon synapse / vesicle docking / regulation of exocytosis / secretion by cell / chloride channel inhibitor activity / SNAP receptor activity / SNARE complex / calcium-ion regulated exocytosis / vesicle fusion / actomyosin / ATP-dependent protein disaggregase activity / hormone secretion / LGI-ADAM interactions / positive regulation of hormone secretion / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / ATP-dependent protein binding / Golgi stack / neurotransmitter secretion / protein localization to membrane / vesicle-fusing ATPase / insulin secretion / syntaxin-1 binding / endosomal transport / Neutrophil degranulation / SNARE complex assembly / positive regulation of neurotransmitter secretion / neurotransmitter transport / regulation of synapse assembly / synaptic vesicle priming / myosin binding / response to gravity / regulation of neuron projection development / positive regulation of receptor recycling / exocytosis / associative learning / modulation of excitatory postsynaptic potential / positive regulation of exocytosis / synaptic vesicle exocytosis / protein sumoylation / synaptic vesicle endocytosis / voltage-gated potassium channel activity / positive regulation of excitatory postsynaptic potential / long-term memory / axonal growth cone / calcium channel inhibitor activity / presynaptic active zone membrane / voltage-gated potassium channel complex / somatodendritic compartment / photoreceptor inner segment / ionotropic glutamate receptor binding / endomembrane system / acrosomal vesicle / axonogenesis / secretory granule / SNARE binding / filopodium / PDZ domain binding / locomotory behavior / intracellular protein transport / trans-Golgi network / postsynaptic density membrane / positive regulation of insulin secretion / potassium ion transport / kinase binding / Schaffer collateral - CA1 synapse / long-term synaptic potentiation / neuron differentiation / terminal bouton / positive regulation of protein catabolic process / calcium-dependent protein binding / synaptic vesicle / synaptic vesicle membrane / actin cytoskeleton / lamellipodium
Similarity search - Function
: / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Vesicle-fusing ATPase / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain ...: / NSF, AAA+ ATPase lid domain / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Vesicle-fusing ATPase / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain / Syntaxin / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / SNARE / Helical region found in SNAREs / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / Vesicle-fusing ATPase / Syntaxin-1A / Synaptosomal-associated protein 25
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsWhite, K.I. / Brunger, A.T.
Funding support United States, 3items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Helen Hay Whitney Foundation United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH063105 United States
Citation
Journal: Nat Commun / Year: 2025
Title: Structural remodeling of target-SNARE protein complexes by NSF enables synaptic transmission.
Authors: K Ian White / Yousuf A Khan / Kangqiang Qiu / Ashwin Balaji / Sergio Couoh-Cardel / Luis Esquivies / Richard A Pfuetzner / Jiajie Diao / Axel T Brunger /
Abstract: Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and ...Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.
#1: Journal: bioRxiv / Year: 2025
Title: Structural remodeling of target-SNARE protein complexes by NSF enables synaptic transmission.
Authors: K Ian White / Yousuf A Khan / Kangqiang Qiu / Ashwin Balaji / Sergio Couoh-Cardel / Luis Esquivies / Richard A Pfuetzner / Jiajie Diao / Axel T Brunger /
Abstract: Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and ...Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.
History
DepositionJun 29, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release
Revision 1.1Oct 8, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Vesicle-fusing ATPase
B: Vesicle-fusing ATPase
C: Vesicle-fusing ATPase
D: Vesicle-fusing ATPase
E: Vesicle-fusing ATPase
F: Vesicle-fusing ATPase
H: Syntaxin-1A
I: Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera
J: Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera
G: Syntaxin-1A
K: Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera
L: Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera
M: Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera
N: Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)915,17427
Polymers909,39214
Non-polymers5,78113
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 14 molecules ABCDEFHGIJKLMN

#1: Protein
Vesicle-fusing ATPase / N-ethylmaleimide-sensitive fusion protein / NEM-sensitive fusion protein / Vesicular-fusion protein NSF


Mass: 82907.430 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: NSF / Production host: Escherichia coli (E. coli) / References: UniProt: P18708, vesicle-fusing ATPase
#2: Protein Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 31045.934 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stx1a, Sap / Production host: Escherichia coli (E. coli) / References: UniProt: P32851
#3: Protein
Synaptosomal-associated protein 25,Synaptosomal-associated protein 25, Alpha-soluble NSF attachment protein chimera / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 58309.328 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Snap25, Snap, Napa, Snap, Snapa / Production host: Escherichia coli (E. coli) / References: UniProt: P60881

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Non-polymers , 3 types, 13 molecules

#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1The 22bin20S complex of NSF, alphaSNAP, and the soluble 2:2 binary SNARE complex of syntaxin-1a and SNAP-25COMPLEX#1-#30MULTIPLE SOURCES
2Subcomplex of alphaSNAP-syntaxin-1a-SNAP-25COMPLEX#2-#31RECOMBINANT
3Homohexameric NSFCOMPLEX#11RECOMBINANT
42:2 binary complex of SNAP-25 and syntaxin-1aCOMPLEX#2-#32RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Rattus norvegicus (Norway rat)10116
43Cricetulus griseus (Chinese hamster)10029
54Rattus norvegicus (Norway rat)10116
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
54Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCl1
2150 mMsodium chlorideNaCl1
31 mMmagnesium chlorideMgCl21
41 mMadenosine triphosphate1
51 mMTCEP1
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 33.96 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1.4particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
5RELION3.1.4CTF correction
8Coot0.9model fitting
9UCSF ChimeraXmodel fitting
11PHENIX1.21model refinement
12RELION3.1.4initial Euler assignment
13cryoSPARC3.2.0final Euler assignment
14RELION3.1.4classification
15cryoSPARC3.2.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1889717
3D reconstructionResolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50776 / Algorithm: EXACT BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6MDM

6mdm


Accession code: 6MDM / Source name: PDB / Type: experimental model

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